Biochemical characterization of rab proteins from Bombyx mori.
The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.
Uno
Tomohide
T
Agrobioscience, Graduate School of Agricultural Science, Kobe University, Hyogo, Japan. unotom@kobe-u.ac.jp
Moriwaki
Tsubasa
T
Nakamura
Masahiko
M
Matsubara
Mamoru
M
Yamagata
Hiroshi
H
Kanamaru
Kengo
K
Takagi
Michihiro
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006868:Hydrolysis; D015151:Immunoblotting; D019476:Insect Proteins / Q000302:isolation & purification / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes; D020691:rab GTP-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D020691:rab GTP-Binding Proteins